Esterase gene and its use

ABSTRACT

An isolated esterase gene coding for an esterase capable of causing asymmetric hydrolysis of an organic carboxylic acid ester of a cyclopentenolone of formula I:  
                 
 
     wherein R 1  is hydrogen or methyl, and R 2  is C 1 -C 10  alkyl, C 2 -C 10  alkenyl, C 2 -C 10  alkynyl, C 1 -C 4  haloalkyl, a C 5 -C 9  aliphatic hydrocarbon moiety which may be optionally protected on the terminal hydroxyl group thereof, or a C 5 -C 9  fatty acid residue which may be optionally protected on the terminal carboxyl group thereof, to produce the cyclopentenolone of formula I in (R)-form, and hybridizing to the base sequence of SEQ ID NO: 1, is useful for the industrially favorable production of optically active cyclopentenolones of formula I which serve as the intermediates of drugs, agricultural chemicals or other active products.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part application of U.S.application Ser. No. 08/978,589, filed Nov. 26, 1997, the contents ofwhich are hereby entirely incorporated by reference.

FIELD OF INVENTION

[0002] The present invention relates to an esterase gene and its use.

BACKGROUND OF THE INVENTION

[0003] Cyclopentenolones of formula I

[0004] wherein R₁ is hydrogen or methyl, and R₂ is C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₄ haloalkyl, a C₅-C₉ hydroxy aliphatichydrocarbon moiety which may be optionally protected on the terminalhydroxyl group thereof, or a C₅-C₉ fatty acid residue which may beoptionally protected on the terminal carboxyl group thereof, areimportant as the intermediates of drugs, agricultural chemicals or otheractive products

[0005] These cyclopentenolones are useful as the important alcoholcomponents in a group of ester compounds, commonly called “syntheticpyrethroids,” having excellent insecticidal activity, and they are alsouseful as the intermediates of prostaglandin derivatives which serve asdrugs.

[0006] For example, the compound of formula II below, au ester of4-hydroxy-3-methyl-2-(2-propynyl)cyclopent-2en-1-one with2,2,3,3-tetramethylcyclopropanecarboxylic acid, is an excellentinsecticide having very strong knockdown activity and mortal activity(see, e.g., JP-B 50-15843/1975).

[0007] The cyclopentenolones of formula I include two kinds of opticalisomers because they have an asymmetric carbon atom at position 4. Inthe case of synthetic pyrethroids containing such optical isomers as thealcohol components, it is well known that the difference in opticalisomerism between these alcohol components makes a great difference intheir insecticidal effects. For example, the compound of formula IIabove has been found to exhibit several times as excellent insecticidalactivity in the case of an ester of(S)-4-hydroxy-3-methyl-2-(2-propynyl)cyclopent-2-en-1-one as in the caseof an ester of the corresponding(R)4-hydroxy-3-methyl-2-(2-propynyl)cyclopent-2-en-1-one.

[0008] Furthermore, similar optically active cyclopentenolones offormula I, such as 4-hydroxy-2-(7-hydroxyheptyl)-2-cyclopentenone,4-hydroxy-2-(6-methoxycarbonylhexyl)-2-cyclopentenone and4hydroxy-2-(2-propenyl)-2-cyclopentenone, are useful as theintermediates of prostaglandin derivatives which serve as drugs.

[0009] For these reasons, there has been a great demand for thedevelopment of a method for separating and obtaining the optical isomersof cyclopentenolones of formula I as the intermediates of drugs,agricultural chemicals or other active products in an industriallyfavorable manner. In addition, for this purpose, in order to prepare amicroorganism, for example, by a gene engineering technique, whichmicroorganism can produce an excellent esterase capable of acting uponan organic carboxylic acid ester of a cyclopentenolone of formula I forasymmetric hydrolysis of the ester, the search of a gene coding for suchan esterase has also been eagerly desired.

SUMMARY OF THE INVENTION

[0010] Under these circumstances, the present inventors have extensivelystudied and found an esterase gene coding for an esterase capable ofacting upon an organic carboxylic acid ester of a cyclopentenolone offormula I for asymmetric hydrolysis of the ester to produce thecyclopentenolone in (R)-form with high optical purity, therebycompleting the present invention.

[0011] Thus, the present invention provides:

[0012] 1) An isolated esterase gene coding for an esterase capable ofcausing asymmetric hydrolysis of an organic carboxylic acid ester of acyclopentenolone of formula I:

[0013] wherein R₁ is hydrogen or methyl, and R₂ is C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₄ haloalkyl, a C₅-C₉ hydroxy aliphatichydrocarbon moiety which may be optionally protected on the terminalhydroxyl group thereof, or a C₅-C₉ fatty acid residue which may beoptionally protected on the terminal carboxyl group thereof, to producethe cyclopentenolone of formula I in (R)-form, and hybridizing to thebase sequence of SEQ ID NO: 1 (hereinafter referred to as the presentgene);

[0014] 2) me isolated esterase gene according to item 1, wherein thehomology of the gene to the base sequence of SEQ ID NO: 1 is 90% orhigher.

[0015] 3) The isolated esterase gene according to item 1, having a basesequence coding for the amino acid sequence of SEQ ID NO: 2.

[0016] 4) The isolated esterase gene according to item 1, having thebase sequence of SEQ ID NO: 1.

[0017] 5) A plasmid containing the isolated esterase gene of item 1, 2,3 or 4 (hereinafter referred to as the present plasmid).

[0018] 6) A transformant obtained by transformation with the plasmid ofitem 5 (hereinafter referred to as the present transformant).

[0019] 7) The transformant according to item 6, which is amicroorganism.

[0020] 8) An esterase produced by a microorganism having the isolatedesterase gene of item 1, 2, 3 or 4 (hereinafter referred to as thepresent esterase).

[0021] 9) The esterase according to item 8, wherein the microorganismhaving the isolated esterase gene of item 1, 2, 3 or 4 is thetransformant of item 6.

[0022] 10) A process for producing en esterase, which comprisescultivating the transformant of item 6 to produce an esterase capable ofcausing asymmetric hydrolysis of an organic carboxylic acid ester of acyclopentenolone of formula I:

[0023] wherein R₁ is hydrogen or methyl, and R₂ is C₁-C₁ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₄ haloalkyl, a C₅-C₉ hydroxy aliphatichydrocarbon moiety which may be optionally protected on the terminalhydroxyl group thereof, or a C₅-C₉ fatty acid residue which may beoptionally protected on the terminal carboxyl group thereof, to producethe cyclopentenolone of formula I in (R)-form (hereinafter referred toas the present production process).

[0024] 11) A method for the optical resolution of a cyclopentenolone offormula I:

[0025] wherein R₁ is hydrogen or methyl, and R₂ is C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₄ haloalkyl, a C₅-C₉ aliphatic hydrocarbonmoiety which may be optionally protected on the terminal hydroxyl groupthereof, or a C₅-C₉ fatty acid residue which may be optionally protectedon the terminal carboxyl group thereof, which comprises allowing theesterase of item 8 to act upon an organic carboxylic acid ester of thecyclopentenolone of formula I for asymmetric hydrolysis of the ester;and separating the cyclopentenolone of formula I in (R)-form from theester of the corresponding enantiomer thereof.

[0026] 12) The optical resolution method according to item 11, whereinthe cyclopentenolone of formula I is4-hydroxy-3-methyl-2-(2-propenyl)cyclopent-2-en-1-one.

[0027] 13) The optical resolution method according to item 11, whereinthe cyclopentenolone of formula I is4hydroxy-3-methyl-2-(2-propynyl)cyclopent-2en-1-one.

BRIEF DESCRIPTION OF THE DRAWING

[0028] The sole Figure is a diagram showing the restriction endonucleasemaps of pAL601 and pAL612, which are specific examples of the presentplasmids.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present gene is an isolated esterase gene coding for anesterase capable of causing asymmetric hydrolysis of an organiccarboxylic acid ester of a cyclopentenolone of formula I to produce thecyclopentenolone of formula I in (R)-form, and hybridizing to the basesequence of SEQ ID NO: 1. The term “esterase” as used herein refers toan esterase as defined in a broad sense containing lipases.

[0030] The phrase “capable of causing asymmetric hydrolysis of anorganic carboxylic acid ester of a cyclopentenolone of formula I toproduce the cyclopentenolone of formula I in (R)-form” as used hereinmeans that an esterase referred to by this phrase can cause asymmetrichydrolysis of an organic carboxylic acid ester of a cyclopentenolone offormula I, such as 4-hydroxy-3-methyl-2-methylcyclopent-2-en-1-one,4-hydroxy-3-methyl-2-ethyl-2-cyclopent-2-en-1-one,4hydroxy-3-methyl-2-(2-propenyl)-2-cyclopent-2-en-1-one,4-hydroxy-3-methyl-2-(2,4-pentadienyl)-2-cyclopent-2en-1-one,4hydroxy-3-methyl-2-(1-methyl-2-propynyl)-2-cyclopent-2-en-1-one,4hydroxy-3-methyl-2-(2-propynyl)cyclopent-2-en-1-one,4-hydroxy-3-methyl-2-(1-methyl-2-propynyl)cyclopent-2en-1-one,4-hydroxy-3-methyl-2-(2,2,2-trifluoroethyl)cyclopent-2-en-1-one,4-hydroxy-2-(7-acetoxyheptyl)-2-cyclopentenone,4-hydroxy-2-(7-hydroxyheptyl)-2-cyclopentenone,4-hydroxy-2-(6-methoxycarbonylhexyl)-2-cyclopentenone or4-hydroxy-2-(2-propenyl)-2-cyclopentenone, to produce the correspondingcyclopentenolone in (R)-form.

[0031] In the cyclopentenolone of formula I, variables have thefollowing meanings.

[0032] The C₁-C₁₀ alkyl represented by R₂ may include, for example,methyl ethyl, pentyl, heptyl and decyl.

[0033] The C₂-C₁₀ alkenyl represented by R₂ may include, for example,2-propenyl, 1-methyl-2-propenyl, 2,4pentadienyl, 2-heptenyl and2-decenyl.

[0034] The C₂-C₁₀ alkynyl represented by R₂ may include, for example,2-propynyl, 1-methyl-2-propynyl, 2-heptynyl and 2-decynyl.

[0035] The C₁-C₄ haloalkyl represented by R₂ may include, for example,2,2,2-tri-fluoroethyl and 4,4,4-trifluorobutyl.

[0036] The C₅-C₉ hydroxy aliphatic hydrocarbon moiety which may beoptionally protected on the terminal hydroxyl group thereof may includeC₅-C₉ hydroxy aliphatic hydrocarbon moieties which may be optionallyprotected on the terminal hydroxy groups thereof with, for example,alkyl or alkoxyalkyl. Particularly preferred are such substituents thataliphatic hydrocarbons contained therein are straight chain saturatedaliphatic hydrocarbons, specific examples of which are 7-acetoxypentyl,7-hydroxypentyl, 7-acetoxyheptyl, 7-hydroxyheptyl and 10-hydroxynonyl.

[0037] The C₅-C₉ fatty acid residue which may be optionally protected onthe terminal hydroxyl group thereof may include C₅-C₉ fatty acidresidues which may be optionally protected on the terminal hydroxylgroup thereof with, for example, alkyl. Particularly preferred are suchsubstituents that fatty acids contained therein are straight chainsaturated fatty acids, specific examples of which are6-methoxycarbonylhexyl and 8-methoxycarbonyloctyl.

[0038] In the above organic carboxylic acid ester, the organiccarboxylic acid may include, for example, C₁-C₁₈ saturated orunsaturated fatty acids, and pyrethroid acids.

[0039] The gene “hybridizing to the base sequence of SEQ ID NO: 1”refers to a gene which can be visually detected by Southernhybridization as described, for example, in “Cloning and Sequence”(complied under the supervision of Itaru Watanabe, edited by MasahiroSugiura, 1989, published by Noson Bunka-sha), using DNA having the basesequence of SEQ ID NO: 1 as a probe. The gene may be DNA having the basesequence of SEQ ID NO: 1 or DNA having a base sequence with theaddition, deletion or replacement of one or more bases in the DNA havingthe base sequence of SEQ ID NO: 1. For example, double-stranded DNA isdissociated into the complementary single-stranded DNAs by heattreatment at 95° C. for 1 minute or by alkali treatment with 0.5 M NaOH,1.5 M NaCl, which are then left cooling on ice for 1 minute or subjectedto neutralization with 0.5 M Tris-HCl (pH 7.0), 3.0 M NaCl, so as toassociate with single-stranded DNA or single-stranded RNA, which iscomplementary to the above single-stranded DNAs, to fall into adouble-stranded state (i.e., hybridized state) again. Such DNA may beusually a gene having a base sequence with a high homology (e.g., about90% or higher homology as a whole, although it may vary depending uponwhether the region is closely related to an active site or a structure)to the base sequence of SEQ ID NO: 1.

[0040] Homologies can be calculated with the homology search programdeveloped by Pearson and Lipman (see, e.g., Pearson and Lipman, (1988),Proc. Natl. Acad. Sci. USA, 85, 2444). They can also be calculated withthis kind of program contained in the Genetyx-Mac (available fromSoftware Kaihatsu). For this purpose, a homology search program (fasta)found in the World Wide Web Service of the DNA Data Bank of Japan (DDBJ)can also be used.

[0041] A more specific example of the present gene is an esterase genehaving a base sequence coding for the amino acid sequence of SEQ ID NO:2. Of course, the present gene may also include an esterase gene havingthe base sequence of SEQ ID NO: 1.

[0042] The present gene can be obtained by the PCR method using genomicDNA prepared, for example, from a microorganism of the genusBurkholderia by an ordinary method (e.g., the method described in “ShinSaibo Kogaku Jikken Protocol” (edited by the Cancer Control ResearchGroup, Medical Science Laboratory, Tokyo University, published byShujun-sha, 1993) as a template and using a fragment of DNA having thebase sequence of SEQ ID NO: 1 (e.g., a combination of about 14 bp ormore oligonucleotides complementary to the 5′-terminal sequence in thebase sequence of SEQ ID NO: 1 and about 14 bp or more oligonucleotidescorresponding to the 3′-terminal sequence in the base sequence of SEQ IDNO: 1; or a combination of about 14 bp or more oligonucleotidescorresponding to the 5′-terminal sequence in the base sequence of SEQ IDNO: 1 and about 14 bp or more oligonucleotides complementary to the3′-terminal sequence in the base sequence of SEQ ID NO: 1) as a primer.

[0043] The present gene can also be obtained by a method such as colonyhybridization or plaque hybridization with a gene library constructed bythe insertion of genomic DNA prepared, for example, from a microorganismof the genus Burkholderia by an ordinary method (e.g., the methoddescribed in “Shin Saibo Kogaku Jikken Protocol” (edited by the CancerControl Research Group, Medical Science Laboratory, Tokyo University,published by Shujun-sha, 1993) into λ phages or plasmids, using a basesequence coding for the amino acid sequence of SEQ ID NO: 2, preferablya 15 bp or more DNA fragment contained in the base sequence of SEQ IDNO: 1, as a probe.

[0044] As the microorganism for use in the preparation of the presentgene, among the microorganisms of the above genus, particularlypreferred is Burkholderia cepacia, a specific example of which isBurkholderia cepacia strain SC-20.

[0045]Burkholderia cepacia strain SC-20 is a microorganism that has beenfound from the nature by the present inventors, and it has thebacteriological characteristics as shown in Table 1. TABLE 1 (1)Morphology Rods (2) Gram staining Negative (3) Spore − (4) Motility +(5) Flagellar arrangement Single polar (6) Attitude to oxygen Aerobic(7) Oxidase test + (8) Catalase test + (9) OF test O (oxidation) (10)Tone of colonies Yellow (11) Fluorescent pigment formation − (12)Water-soluble pigment formation + (13) PHB accumulation + (14)Protocatechuate cleavage ortho (15) Arginine dehydrolase − (16) Growthat 40° C. + (17) Denitrification − (18) Nitrate reduction − (19) Gelatinliquefaction + (20) Starch hydrolysis − (21) Utilization of: (a)Glucose + (b) Xylose + (c) Ribose + (d) Rhamnose + (e) Levulinate + (f)Mesaconate − (g) D-Tartrate − (h) 2,3-Butylene glycol + (i) Tryptamine −(22) Quinone type Q-8 (23) GC content of DNA 68 in bacterial cells (mol%)

[0046] These bacteriological characteristics are found to be consistentwith those of Burkholderia cepacia when compared with the data inBergey's Manual of Systematic Bacteriology, Vol. 1 (1984); Bergey'sManual of Determinative Bacteriology, Ninth edition (1994); Zhao et al.,Int. J. Syst. Bacteriol., 45, p. 600 (1995); and Yabuuchi et al.,Microbiol. Immnunol., 36, p. 1251 (1992).

[0047] The present gene can be obtained by the PCR method using DNAprepared from the bacterial cells of Escherichia coli strainJM109/pAL612 as a template and using a fragment of DNA having the basesequence of SEQ ID NO: 1 (e.g., a combination of about 14 bp or moreoligonucleotides complementary to the 5′-terminal sequence in the basesequence of SEQ ID NO: 1 and about 14 bp or more oligonucleotidescorresponding to the 3′-terminal sequence in the base sequence of SEQ IDNO: 1; or a combination of about 14 bp or more oligonucleotidescorresponding to the 5′-terminal sequence in the base sequence of SEQ I)NO: 1 and about 14 bp or more oligonucleotides complementary to the3′-terminal sequence in the base sequence of SEQ ID NO: 1) as a primer.

[0048]E. coli strain JM109/pAL612 is a transformant microorganismobtained by the incorporation of plasmid pAL612 containing the presentgene (the present plasmid) into E. coli strain JM109 (the presenttransformant), and it has been deposited in the National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology as “FERM-BP 5740”(accession date: Nov. 7, 1996).

[0049] The present plasmid can be easily constructed by incorporatingthe present gene obtained, for example, into a vector which has beenusually used in host cells to be transformed, by an ordinary geneengineering technique. More specifically, for example, when E. coli as amicroorganism is used as the host cell, the vector to be used mayinclude pUC119 (available from Takara Shuzo) and pBluescriptII(available from Stratagene Cloning System).

[0050] The method for transforming a host cell with the present plasmidconstructed may be a method usually used depending upon the host cell tobe transformed, and for example, when E. coli as a microorganism is usedas the host cell, it may include an ordinary method as described in“Molecular Cloning” (J. Sambrook et al., Cold Spring Harbor, 1989).

[0051] The selection of transformants is carried out as follows: Forexample, the host cell transformed with the present plasmid is firstcultivated on an LB plate containing tributyrin, and those forming aclear zone are selected. The selected transformants are cultivated, andthe resulting cultures are treated with an organic carboxylic acid esterof a cyclopentenolone of formula I. The reaction products are analyzed,so that transformants producing the cyclopentenolone of formula I in(R)-form with high optical purity may be selected.

[0052] More specifically, for example, 0.5 g of the acetic acid ester of(RS)-4-hydroxy-3-methyl-2-(2-propenyl)cyclopent-2-en-1-one and 8.0 ml of50 mM phosphate buffer (pH 7.0) are put into a 100 ml sample bottle, andthe mixture is preheated at 40° C. under stirring with a stirring barfor 10 minutes. To this mixture is added 1.0 ml of the above culture,and the reaction is effected at 40° C. under stirring with a stirringbar. After 30 minutes, the reaction mixture is taken in a volume of 50μl, and the reaction is stopped by the addition of 1 ml of ethanol. Forblanks, purified water is used instead of the culture, and the test isconducted in the same manner. The rate of decomposition is determined bygas chromatography. As the column for analysis, 10% silicone DC-QF-1,2.6 m long, is used, and the analysis is carried out with GC-14A(available from Shimazu Seisakusho) under the following conditions:column temperature, 150° C.; injection temperature, 170° C.; detectiontemperature, 170° C.; and detector, FID. For the enzyme titer, theamount of enzyme releasing 1 μmol of (R)-4hydroxy-3-methyl-2-(2-propenyl)-cyclopent-2-en-1-one for 1 minute isdefined as 1 unit. The reaction mixture is further extracted with methylisobutyl ketone, and the extract is examined for the optical purity of(R)-4hydroxy-3-methyl-2-(2-propenyl)cyclopent-2-en-1-one by the HPLCanalysis. In the analysis, columns for optical isomer analysis OA4100(4.0 mm I.D.×25 cm) available from Sumika Bunseki Center are used. Asthe eluent, a mixture of hexane, 1,2-dichloroethane and ethanol at aratio of 100:20:1 can be used. The optical isomer ratio may bedetermined at a flow rate of 1.0 ml/min. with an absorbance at 230 nm asan index.

[0053] More particularly, from the selected transformants, plasmidscontained in the transformants are prepared, and the restrictionendonuclease maps of the plasmids thus prepared are constructed by anordinary method as described, for example, in “Molecular Cloning” (J.Sambrook et al., Cold Spring Harbor, 1989). It can also be determinedwhether the desired present gene is contained or not by a method such asbase sequence analysis, Southern hybridization or Western hybridization.

[0054] In this manner, the present transformants can be obtained andcultivated to produce the present esterase (the present productionprocess).

[0055] When the transformants are microorganisms, the transformants arecultivated with various kinds of media suitably containing carbonsources, nitrogen sources, organic salts and/or inorganic salts, andother additives, which have been used for preparing the ordinarycultures of microorganisms. The carbon sources may include glucose,glycerol, dextrin, sucrose, organic acids, animal and vegetable oils,and molasses. The nitrogen sources may include organic and inorganicnitrogen sources such as broth, peptone, yeast extract, malt extract,soy bean powder, corn steep liquor, cotton seed powder, dry yeast,casamino acid, sodium nitrate and urea. The organic and inorganic saltsmay include chlorides, sulfates, acetates, carbonates and phosphates ofelements such as potassium, sodium, magnesium, iron, manganese, cobaltand zinc, specific examples of which are sodium chloride, potassiumchloride, magnesium sulfate, ferrous sulfate, manganese sulfate, cobaltchloride, zinc sulfate, copper sulfate, sodium acetate, calciumcarbonate, sodium carbonate, potassium monohydrogenphosphate andpotassium dihydrogenphosphate.

[0056] Furthermore, the addition of triglycerides such as olive oil tothe medium is preferred. The amount of triglycerides to be added may beabout 10 mg to about 10 g for each 100 ml of the medium.

[0057] Cultures are prepared by an ordinary method for microorganisms,and they can be in the form of either solid cultures or liquid cultures(e.g., shaking cultures using test tubes or reciprocating shakers, andother cultures using jar fermenters or fermentation tanks). Inparticular, when jar fermenters are used, it is necessary to introduceaseptic air thereinto, usually at a rate of about 0.1 to about 2 timesthe culture volume per minute. The incubation temperature may besuitably altered within a range to ensure the growth of microorganisms.For example, cultures are preferably incubated at a temperature of about15° C. to about 40° C. under the control of medium pH within the rangeof about 6.0 to about 8.0. The incubation period may vary on variousconditions of incubation, and the preferred incubation period is usuallyin the range of about 1 to about 5 days.

[0058] The present esterase has the following characteristics:

[0059] 1) The molecular weight (determined by SDS-PAGE) is about 38kilo-daltons;

[0060] 2) The isoelectric point (pI) is 6.0;

[0061] 3) The optimum temperature is about 60° C., and the reaction canbe effected in the range of at least about 20° C. to about 70° C.,preferably about 30° C. to about 40° C.;

[0062] 4) The reaction can be effected in the pH range of about 4 toabout 9, preferably about 5 to about 7;

[0063] 5) It is capable of causing asymmetric hydrolysis of an organiccarboxylic acid ester of a cyclopentenolone of formula I to produce thecyclopentenolone of formula I in (R)-form;

[0064] 6) It can also be obtained by cultivating non-transformants, forexample, of a microorganism of the genus Burkholderia (particularlypreferred is Burkholderia cepacia, a specific example of which isBurkholderia cepacia stain SC-20). Of course, as described above, it canalso be obtained by cultivating the transformants which have beenprepared by transformation with a plasmid containing the present gene.

[0065] The present esterase may be utilized for the enzyme reaction inthe form of a culture containing the same, but may also be utilized forthe enzyme reaction in the form of a crude enzyme separated from theculture or in the form of a purified enzyme. The crude enzyme may beseparated by an ordinary method, for example, in which bacterial cellsare disrupted by ultrasonic disintegration, trituration with glass beadsor alumina, homogenization or disruption with a French press, enzymetreatment with lysozyme, and the desired fraction is obtained from thedisrupted bacterial cells by salt deposition with ammonium sulfate orany other salt; precipitation with an organic solvent or an organicpolymer such as polyethylene glycol; chromatography such as ion exchangechromatography, hydrophobic chromatography, gel filtrationchromatography, affinity chromatography or any other chromatography; orelectrophoresis. If necessary, these techniques can be used incombination.

[0066] Furthermore, the present esterase can also be utilized for theenzyme reaction in the form of an immobilized product which has beenobtained by insolubilizing the esterase by a method of immobilization,such as a carrier binding technique in which the esterase is attached toa carrier by covalent bonding, ion bonding or absorption; or anentrapment technique in which the esterase is entrapped into the networkstructure of a polymer; and then by processing the insolubilizedesterase into an easily separable state.

[0067] The present esterase can be utilized, for example, in the opticalresolution of a cyclopentenolone of formula I. That is, the presentesterase can be allowed to act upon an organic carboxylic acid ester ofa cyclopentenolone of formula I for asymmetric hydrolysis of the ester,so that the cyclopentenolone of formula I in (R)-form is separated fromthe ester of the corresponding enantiomer thereof in (S)-form. In such aresolution, esters in racemic form are usually used as the startingmaterial.

[0068] Specific examples of the cyclopentenolone of formula I are4hydroxy-3 -methyl-2-(2-propenyl)cyclopent-2-en-1-one,4hydroxy-3-methyl-2-(2-propynyl)cyclopent-2-en-1-one,4-hydroxy-2-(7-hydroxyheptyl)-2-cyclopentenone,4-hydroxy-2-(6-methoxycarbonylhexyl)-2-cyclopentenone and4-hydroxy-2-(2-propenyl)-2-cyclopentenone.

[0069] The reaction temperature is, for example, in the range of about20° C. to about 70° C., preferably about 30° C. to about 40° C. Thereaction pH is, for example, in the range of about 4 to about 9,preferably about 5 to about 7. The reaction time is, for example, in therange of about 5 minutes to about 96 hours.

[0070] The cyclopentenolone of formula I in (R)-form and the ester ofthe corresponding enantiomer thereof can be recovered from the reactionmixture by any method generally known in the art. For example,procedures such as extraction with a solvent, fractional distillationand column chromatography can be suitably employed. More specifically,the reaction mixture is extracted with an organic solvent such as ether,ethyl acetate or benzene, and the extract is subjected to factionaldistillation, or to silica gel chromatography, followed by extraction,so that the cyclopentenolone of formula I in (R)-form is separated fromthe ester of the corresponding enantiomer thereof. Thus, the desiredsynthetic pyrethroids or prostaglandin derivatives can be obtained fromthe separated products.

[0071] The cyclopentenolone of formula I in (R)-form thus recovered canbe easily converted into the cyclopentenolone of formula I in (S)-form,which is important as the alcohol intermediate of synthetic pyrethroidsor as the intermediate of prostaglandin derivatives, by directhydrolysis or by hydrolysis after tosylation or mesylation according toany one of the methods described in JP-A 52-156840/1977, JP-B4-5019/1992 and JP-B 5-36429/1993 depending upon the purpose of use, foroptical reversal to change into the corresponding enantiomer. Thecyclopentenolone thus obtained can be used, for example, byesterification, to produce the desired synthetic pyrethroids orprostaglandin derivatives.

[0072] The ester in (S)-form remaining after the asymmetric hydrolysiscan be converted into the cyclopentenolone of formula I in (S)-form bydirect hydrolysis, or by hydrolysis after the pre-hydrolysis or afterthe tosylation or mesylation of the reaction products of the asymmetrichydrolysis as described in JP-B 5-79656/1993. The cyclopentenolone offormula I in (S)-form can be used, for example, by esterification in thesame manner as described above, to produce the desired syntheticpyrethroids or prostaglandin derivatives.

[0073] Thus, according to the present invention, the organic carboxylicacid esters of cyclopentenolones of formula I can also be entirelyconverted into the organic carboxylic acid esters of cyclopentenolonesof formula I in (S)-form; the present production process is, therefore,extremely effective from an industrial point of view.

EXAMPLES

[0074] The present invention will be further illustrated by thefollowing examples; however, the present invention is not limited tothese examples in any way whatsoever.

Example 1 Preparation of Genomic DNA

[0075] A culture of Burkholderia cepacia strain SC-20 was grown on amedium (Bacto tryptone (available from Difco Laboratories Incorporated),10 g; Bacto yeast extract (available from Difco LaboratoriesIncorporated), 5 g; NaCl, 5 g/l; hereinafter referred to simply as LBmedium) at 30° C. for 12 hours, and then harvested by centrifugation at6000 rpm for 10 minutes to collect the bacterial cells.

[0076] The collected bacterial cells were suspended in a solution (10 mMTris-HCl (pH 8.0), 1 mM EDTA-NaOH (pH 8.0), 10 mM NaCl; hereinafterreferred to simply as TEN solution) containing 1 mg/ml lysozyme chloride(available from Seikagaku Kogyo) and 25 μg/ml RNaseA (available fromSigma Aldrich Japan), and then incubated at 37° C. for 20 minutes.Thereafter, sodium dodecylsulfate was added to a final concentration of1% (w/v), and incubation was continued at 55° C. for 10 minutes. Then,phenol saturated with TE [10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)]was added in the same volume. The mixture was slowly stirred and thencentrifuged at 10,000 rpm for 10 minutes to collect the upper layer, towhich a TE-saturated phenol-chloroform solution was added in the samevolume. The mixture was slowly stirred and then centrifuged at 10,000rpm for 10 minutes to collect the upper layer, to which a 3M ammoniumacetate solution was added in a 1/10-fold volume and then ethanol in a2-fold volume. The DNA being deposited was taken by rolling it up rounda glass rod. This DNA was rinsed with 70% (v/v) ethanol, and then rinsedagain with 80% (v/v) ethanol and 100% ethanol, successively, followed byair drying. The DNA thus obtained was suspended in a TEN solutioncontaining 25 μg/ml RNaseA (available from Sigma Aldrich Japan) and 20μg/ml Proteinase K (available from Boehringer Manaheim), and thenincubated at 37° C. for 12 hours, to which a TE-saturatedphenol-chloroform solution was added in the same volume. The mixture wasslowly stirred and then centrifuged at 10,000 rpm for 10 minutes tocollect the upper layer, to which a 3M ammonium acetate solution wasadded in a 1/10-fold volume and then ethanol, in a 2-fold volume. TheDNA being deposited was taken by rolling it up round a glass rod. ThisDNA was rinsed with 70% (v/v) ethanol, and then rinsed again with 80%(v/v) ethanol and 100% ethanol, successively, followed by air drying.The DNA thus obtained was dissolved in 10 ml of TE solution containing25 μg/ml RNaseA, and the solution was dialyzed twice against 2L of TEsolution. Thus, about 1.6 mg of genomic DNA was obtained from 100 ml ofthe culture.

Example 2 Preparation of Genomic DNA Library

[0077] Fifty micrograms of the genomic DNA obtained above was digestedwith the restriction endonuclease Eco RI at 37° C. for 1 hour, followedby agarose gel electrophoresis (0.7% concentration). DNA fractionscorresponding to the size of 9.4 kb to 6.6 kb and those corresponding tothe size of 7.5 kb to 5.5 kb were cut out of the agarose gel, and thenpurified with Gene Clean (available from BIO101).

[0078] The purified DNA fragments and λZAPII DNA (available fromStratagene Cloning System) were ligated together with DNA ligaseaccording to the manual attached to the kit. The ligated DNA was invitro packaged into the λ particles using Gigapack Gold Packaging Kit(available from Stratagene Cloning System) according to the mnanualattached to the kit.

Example 3 Preparation of Probe DNA

[0079] (1) Purification of esterase enzyme

[0080] A culture of Burkholderia cepacia strain SC-20 isolated from thesoil was grown on a medium (soy bean powder extract, 100 ml; corn steepliquor, 1 g; soy bean oil, 5 g) at 30° C. for 48 hours. The soy beanpowder extract was prepared by adding 250 ml of 0.3% NaOH to 10 g of soybean powder, heating the mixture at 70° C. for 1 hour, and filtering themixture through a filter paper. After completion of the cultivation, thesupernatant was collected by centrifugation at 14,000 rpm for 20minutes. To the collected supernatant, ethanol was added to aconcentration of 80%, and the precipitate was collected by furthercentrifugation at 14,000 rpm for 20 minutes.

[0081] The precipitate after drying was suspended in 50 mM phosphatebuffer (pH 7.0), and fractions precipitating in an ammonium sulfateconcentration of 20% to 60% were collected.

[0082] The collected fractions were suspended in 5 ml of 20 mM Tris-HCl(pH 8.0) solution, and the suspension was charged on a DEAE-SepharoseFast Flow column (2.6×5 cm, available from Pharmacia Biotech)equilibrated with 20 mM Tris-HCl (pH 8.0) solution. The column wasthoroughly washed with 20 mM Tris-HCl (pH 8.0) solution. After thewashing, the desired protein was eluted by a gradient of NaClconcentration ranging from 0 M to 0.5 M, resulting in an activefraction. The active fraction obtained was purified in the same manneras described above using a DEAE-Sepharose Fast Flow column (2.6×5 cm,available from Pharmacia Biotech) equilibrated with 10 mM Tris-HCl (pH7.5) solution, resulting in an active fraction. The active fractionobtained was purified by gel filtration chromatography using a Superose12 HR10/30 column (available from Pharmacia) equilibrated with 50 mMphosphate buffer (pH 7.5) containing 150 mM NaCl.

[0083] The active fraction thus obtained (i.e., main peak) was capableof causing asymmetric hydrolysis of the acetic acid ester of(RS)-4-hydroxy-3-methyl-2-(2-propenyl)-cyclopent-2-en-1-one to produce(R)4hydroxy-3-methyl-2-(2-propenyl)cyclopent-2-en-1-one. The activefraction was further capable of causing asymmetric hydrolysis of theacetic acid ester of(RS)-4-hydroxy-3-methyl-2-(2-propynyl)cyclopent-2-en-1-one to produce(R)4hydroxy-3-methyl-2-(2-propynyl)cyclopent2-en-1-one. The analysis ofthe active fraction by SDS electrophoresis [using AutomaticElectrophoresis Apparatus Phast System (available from PharmaciaBiotech) with Phast Gel Gradient 10-15 and Phast Gel SDS buffer strip]revealed that the active fraction is in a single state as a protein andthat the protein is composed of a single subunit and has a molecularweight of about 38 kilo-daltons.

[0084] (2) Preparation of probe DNA

[0085] The protein thus obtained was concentrated by ultrafiltration(available from Grace Japan) with a cut away molecular weight of 10kilo-daltons, desalted on a desalting column (Fast Desalting Column,available from Pharmacia Biotech) equilibrated with water, and thenconcentrated under reduced pressure. The protein thus concentrated wasdigested from the N-terminus using Protein Sequencer 470A (availablefrom Perkin Elmer Japan), and then analyzed with PTH-Analyzer 120A(available from Perkin Elmer Japan) to determine the N-terminal aminoacid sequence. The amino acid sequence determined is shown by SEQ ID NO:3 in the Sequence Listing.

[0086] Based on the N-terminal amino acid sequence determined asdescribed above, an oligonucleotide having the base sequence shown bySEQ ID NO: 4 in the Sequence Listing was synthesized. For the synthesisof the oligonucleotide, DNA automatic synthesizer model 380A (availablefrom Perkin Elmer Japan) was used. The synthesized DNA was labeled witha radioisotope using MEGALABEL kit (available from Takara Shuzo).

Example 4 Isolation of Phage Containing the Present Gene

[0087] The genomic DNA library prepared in Example 2 was screened forthe present gene with Colony/Plaque Screen (available from NEN ResearchProducts) according to the manual attached to the kit. Morespecifically, a culture of E. coli XL1-Blue was grown on an LB medium,and then harvested by centrifugation at 8000 rpm for 15 minutes tocollect the bacterial cells. The bacterial cells collected weresuspended in 10 mM MgSO₄ solution, and the bacterial suspension was theninfected with λ phage containing the genomic DNA library prepared inExample 2. After the infection, the suspension was mixed with a medium(NaCl, 5 g/l; MgSO₄-H₂O, 2 g/l; Bacto yeast extract (available fromDifco Laboratories Incorporated), 5 g/l; NZ amine, 10 g/l; herein-afterreferred to simply as NZY medium) containing 0.7% agarose and warmed to50° C. The mixture was layered on an NZY agar plate of 150 mm indiameter. About twenty thousand phages were placed on each plate, andincubated at 37° C. until plaques appeared. After the incubation, theplaques formed on the plates were transferred to a Colony/Plaque Screenmembrane at a ratio of two membranes per plate. The Colony/Plaque Screenmembranes were treated twice with 0.5 M NaOH solution for 5 minutes,neutralized twice with 1.0 M Tris-HCl (pH 8.0) solution for 5 minutes,and then rinsed with 2×SSC solution, followed by air drying on filterpaper. (The term “n×SSC solution” refers to a n/20-fold dilution of20×SSC solution, and the “20×SSC solution” contains 175.3 g of NaCl and88.2 g of trisodium citrate per liter (pH 7.0); hereinafter referred tosimply as “n×SSC”).

[0088] The Colony/Plaque Screen membranes obtained were immersed in2×SSC, and then immersed in a prehybridization solution warmed to 68° C.for 3 hours, containing 6×SSC, 0.5% (w/v) sodium dodecyl sulfate(hereinafter referred to simply as “SDS”), 5×Denhart's solution (0.1 gof Ficoll, 0.1 g of polyvinylpyrrolidone, and 0.1 g of bovine serumalbumin per 100 ml), and 100 μg/ml denatured sonicated sermon sperm DNA.The membranes thus pretreated were then immersed in a hybridizationsolution containing 6×SSC, 0.5% (w/v) SDS, and 100 μg/ml denaturedsonicated sermon sperm DNA, to which the probe DNA prepared in Example 3was added, and the mixture was left under shaking at 63° C. for 18hours. Thereafter, the membranes were washed with 2×SSC, 0.5% SDSsolution at room temperature for 5 minutes. The membranes were further(1) washed under shaking with 2×SSC, 0.1% SDS solution at roomtemperature for 15 minutes, (2) washed under shaking with 0.1×SSC, 0.5%SDS solution at 37° C. for 60 minutes, and (3) washed under shaking with0.1×SSC, 0.5% SDS solution at 68° C. for 60 minutes to remove excessiveprobe DNA. From the positions corresponding to the probe DNA adsorbed onthe membranes, phases were taken by suction with a Pasteur pipette. Theabove procedures were repeated until the probe DNA was separated as aunique plaque, and phages containing the desired DNA fragment (thepresent gene) were isolated. Thus, about forty thousand plaques werescreened, and three strains of phages were isolated.

Example 5 Acquisition of Transformant and Analysis of Base Sequence

[0089] The phages obtained in Example 4 were recombined into a plasmidaccording to the manual of λ ZAPII (available from Stratagene CloningSystem). The resulting plasmid was designated pAL601. The analysis ofpAL601 with restriction endonucleases revealed that about 7.0 kbp Eco RIfragment was inserted (see FIG. 1).

[0090] For about 2 kbp Sma I fragment (i.e., coding region for theesterase gene) of pAL601, the base sequence was determined with PRISMkit and automatic base sequence analyzer 373A (both available fromPerkin Elmer Japan). The analysis was carried out with Genetyx-Mac/ATSQand Genetyx-Mac (both available from Software Kaihatsu). The basesequence of the esterase gene is shown by SEQ ID NO: 1 in the SequenceListing.

[0091] In the amino acid sequence (shown by SEQ ID NO: 2 in the SequenceListing) deduced from the base sequence determined, there is a regioncompletely corresponding to the amino acid sequence of SEQ ID NO.3, andthe presence of the desired esterase gene on the above DNA fragment wasconfirmed.

Example 6 Optical Selectivity I of Esterase

[0092] pAL601 was digested with the restriction endonuclease Sma I andsubcloned to give pAL612. The resulting pAL612 was transformed into E.coli strain JM109.

[0093] A culture of the transformant E. coli strain JM109/pAL612 thusobtained was grown on 100 ml of LB medium containing 50 mg/l ofampicillin and 50 mg/l of 1 mM isopropyl thio-β-D-galactoside(hereinafter referred to simply IPTG) at 37° C. for 16 hours, and thenharvested by centrifugation at 6000 rpm for 10 minutes to collect thebacterial cell.

[0094] The bacterial cells obtained were suspended in 20 ml of 200 mMphosphate buffer. Then, 0.5 g of the methyl ester of(RS)4-hydroxy-3-methyl-2-(2-propenyl)cyclopent-2-en-1-one and 8.0 ml of50 mM phosphate buffer (pH 7.0) were put in a 100 ml sample bottle, andpreheated under stirring with a stirring bar at 40° C. for 10 minutes.To this mixture was added 1.0 ml of the above suspension, and thereaction was effected at 40° C. under stirring with a stirring bar.After 30 minutes, the reaction mixture was taken in a volume of 50 μl,and the reaction was stopped by the addition of 1 ml of ethanol. Forblanks, purified water was used instead of the culture, and the test wasconducted in the same manner. The rate of decomposition was determinedby gas chromatography. As the column for analysis, 10% silicone DC-QF-1,2.6 m long, was used, and the analysis was carried out with GC-14A (fromShimazu Seisakusho) under the following conditions: column temperature,150° C.; injection temperature, 170° C.; detection temperature, 170° C.;and detector, FID. The reaction mixture was further extracted withmethyl isobutyl ketone, and the extract was examined for optical purityby the HPLC analysis. In the analysis, a column for optical isomeranalysis OA-4100 (4.0 mm I.D.×25 cm) available from Sumika BunsekiCenter was used. As the eluent, a mixture of hexane, 1,2-dichloroethaneand ethanol at a ratio of 100:20:1 was used. The optical isomer ratiowas determined at a flow rate of 1.0 ml/min. with an absorbance at 230nm as an index.

[0095] Based on the results of the above analysis, the rate ofhydrolysis and optical selectivity were calculated and are shown inTable 2. TABLE 2 Optical isomer ratio [(S)/(R)] of released4-hydroxy-3-methyl- Rate of hydrolysis 2-(2-propenyl)cyclopent-Transformant (%) 2-en-1-one JM109/pAL612 45 2/98

[0096] As can be seen from Table 3, E. coli strain JM109/pAL612, whichis a transformant containing the inserted 2 kbp Sma I fragment, producesan esterase capable of causing asymmetric hydrolysis of the acetic acidester of (RS)4hydroxy-3-methyl-2-(2-propenyl)cyclopent-2en-1-one toproduce (R)4hydroxy-3-methyl-2-(2-propenyl)cyclo-pent-2-en-1-one.

Example 7 Optical Selectivity II of Esterase

[0097] The acetic acid ester of(RS)4hydroxy-3-methyl-2-(2-propynyl)cyclopent-2-en-1-one was used as thesubstrate, and the same experiments as described in Example 4 were made.The results are shown in Table 3. TABLE 3 Optical isomer ratio [(S)/(R)]of released 4-hydroxy-3-methyl- Rate of hydrolysis2-(2-propynyl)cyclopent- Transformant (%) 2-en-1-one JM109/pAL612 402/98

Example 8 Optical Selectivity III of Esterase

[0098] The acetic acid diester of(RS)-4hydroxy-2-(7-hydroxyheptyl)-2-cyclopentenone was used as thesubstrate, and the same experiments as described in Example 4 were made.The results are shown in Table 4. As the buffer, 100 mM phosphate buffer(pH 6.0) was used. TABLE 4 Optical isomer ratio [(S)/(R)] of released4-hydroxy- Rate of hydrolysis 2-(7-hydroxyheptyl)- Transformant (%)2-cyclopentenone JM109/pAL612 24 0/80

[0099] The rate of decomposition was determined by gas chromatographyfor the acetic acid diester of4hydroxy-2-(7-hydroxyheptyl)-2-cyclopentenone. As the column foranalysis, a 10% silicone DC-QF-1, 2.6 m long,. was used, and theanalysis was carried out under the following conditions: columntemperature, 240° C. and injection temperature, 260° C. The reactionmixture was further extracted with methyl isobutyl ketone, and theextract was examined for optical purity by the HPLC analysis. In theanalysis of 4hydroxy-2-(7-hydroxyheptyl)-2-cyclopentenone, two columnsfor optical isomer analysis OA-4500 available from Sumika Bunseki Centerwere used in connected form. As the eluent, a mixture of hexane,1,2-dichloroethane and ethanol at a ratio of 100:4:4 was used. Theoptical isomer ratio was determined at a flow rate of 1.5 ml/min. withan absorbance at 235 nm as an index.

Example 9 Optical Selectivity IV of Esterase

[0100] The acetic acid ester of(RS)4hydroxy-2-(6-methoxycarbonylhexyl)-2-cyclopentenone was used as thesubstrate, and the same experiments as described in Example 4 were made.The results are shown in Table 5. TABLE 5 Optical isomer ratio [(S)/(R)]of released 4-hydroxy- Rate of hydrolysis 2-(6-methoxycarbonylhexyl)-Transformant (%) 2-cyclopentenone JM109/pAL612 30 9/91

[0101] The rate of decomposition was determined by gas chromatography.As the column for analysis, 10% silicone DC-QF-1, 2.6 m long, was used,and the analysis was carried out under the following conditions: columntemperature, 240° C. and injection temperature, 260° C. The reactionmixture was further extracted with methyl isobutyl ketone, and theextract was examined for optical purity by the HPLC analysis. In theanalysis of 4-hydroxy-2-(6-methoxycarbonylhexyl)-2-cyclopentenone, acolumn for optical isomer analysis OA4500 available from Sumika BunsekiCenter was used. As the eluent, a mixture of hexane, 1,2-dichloroethaneand ethanol at a ratio of 100:20:1 was used. The optical isomer ratiowas determined at a flow rate of 1 ml/min. with an absorbance at 235 nmas an index.

[0102] As described above, the present invention made it possible toprovide a gene coding for an esterase capable of acting upon an organiccarboxylic acid ester of a cyclopentenolone of formula I for asymmetrichydrolysis of the ester to produce the cyclopentenolone of formula I in(R)-form with high optical purity.

1 4 1 1089 DNA Burkholderia cepacia 1 atgagcagat cgatacgagc gaaggcagtggcgaccgtgg tggcgatcaa cgcggccccg 60 gccgcgagtg ttggaaccgt tctggccatgtcgctggccg gcgcacaggc cgcttccgcc 120 gcgacgaccg ccgttgacga ctacgcggcgacccggtacc cgatcattct cgtgcacggg 180 ctgaccggca ccgacaagta cggtggcgtcgtcgagtact ggtatcgcat tccggaggac 240 ctgcgggcgc acggcgcggc ggtatacgttgccaacctgt ccggcttcca gagcgacgat 300 ggcccgaacg ggcgtggcga gcaattgcttgcattcgtga agcaggtgct cgcggcgacg 360 ggcgcgcaga aggtgaatct gatcggccatagccagggcg gcctgacatc gcgttatgtt 420 gcgtccgttg caccggaact ggtcgcatcggtgacgacga tcagtacgcc gcactggggc 480 tcgcaattcg cggacttcgt ccagcaactgttgcagacgg acccgaccgg cctgtcgtcg 540 accgtgctcg gcgcattcgc gaatgcgctcggcacgttga cgagcagcaa cttcaatacg 600 aaccagaatg cgattcaggc gttgtcggtgctgacgacgg caaaggccgc cgcatacaac 660 cagaaattcc cgagcgccgg tctcggtgcgccgggctcgt gtcaaaccgg cgcgccaacg 720 gagactgtcg gcggcaatac gcatctgctttattcgtggg gcggcacggc gatccagccg 780 acagcgacgg tggccggcgt gacaggggccgtcgatacga gcgtgagcgg ggtcaccgat 840 ccggcgaacg cgctcgatcc gtcaacgctggcactcctcg gcagcggcac ggtgatgatc 900 aatcgcagcg ccggtccgaa cgatggcgtcgtgtcgcaat gcagcgcgcg gtttggccag 960 gtgctcggca cgtatcactg gaatcacaccgatgcgatca accagatcct cggcgtgctc 1020 ggcgcgaatg tggaggatcc ggttgcggtaatccgcacgc acgcgaaccg gttgaagaat 1080 caaggcgtg 1089 2 363 PRTBurkholderia cepacia 2 Met Ser Arg Ser Ile Arg Ala Lys Ala Val Ala ThrVal Val Ala Ile 1 5 10 15 Asn Ala Ala Pro Ala Ala Ser Val Gly Thr ValLeu Ala Met Ser Leu 20 25 30 Ala Gly Ala Gln Ala Ala Ser Ala Ala Thr ThrAla Val Asp Asp Tyr 35 40 45 Ala Ala Thr Arg Tyr Pro Ile Ile Leu Val HisGly Leu Thr Gly Thr 50 55 60 Asp Lys Tyr Gly Gly Val Val Glu Tyr Trp TyrArg Ile Pro Glu Asp 65 70 75 80 Leu Arg Ala His Gly Ala Ala Val Tyr ValAla Asn Leu Ser Gly Phe 85 90 95 Gln Ser Asp Asp Gly Pro Asn Gly Arg GlyGlu Gln Leu Leu Ala Phe 100 105 110 Val Lys Gln Val Leu Ala Ala Thr GlyAla Gln Lys Val Asn Leu Ile 115 120 125 Gly His Ser Gln Gly Gly Leu ThrSer Arg Tyr Val Ala Ser Val Ala 130 135 140 Pro Glu Leu Val Ala Ser ValThr Thr Ile Ser Thr Pro His Trp Gly 145 150 155 160 Ser Gln Phe Ala AspPhe Val Gln Gln Leu Leu Gln Thr Asp Pro Thr 165 170 175 Gly Leu Ser SerThr Val Leu Gly Ala Phe Ala Asn Ala Leu Gly Thr 180 185 190 Leu Thr SerSer Asn Phe Asn Thr Asn Gln Asn Ala Ile Gln Ala Leu 195 200 205 Ser ValLeu Thr Thr Ala Lys Ala Ala Ala Tyr Asn Gln Lys Phe Pro 210 215 220 SerAla Gly Leu Gly Ala Pro Gly Ser Cys Gln Thr Gly Ala Pro Thr 225 230 235240 Glu Thr Val Gly Gly Asn Thr His Leu Leu Tyr Ser Trp Gly Gly Thr 245250 255 Ala Ile Gln Pro Thr Ala Thr Val Ala Gly Val Thr Gly Ala Val Asp260 265 270 Thr Ser Val Ser Gly Val Thr Asp Pro Ala Asn Ala Leu Asp ProSer 275 280 285 Thr Leu Ala Leu Leu Gly Ser Gly Thr Val Met Ile Asn ArgSer Ala 290 295 300 Gly Pro Asn Asp Gly Val Val Ser Gln Cys Ser Ala ArgPhe Gly Gln 305 310 315 320 Val Leu Gly Thr Tyr His Trp Asn His Thr AspAla Ile Asn Gln Ile 325 330 335 Leu Gly Val Leu Gly Ala Asn Val Glu AspPro Val Ala Val Ile Arg 340 345 350 Thr His Ala Asn Arg Leu Lys Asn GlnGly Val 355 360 3 15 PRT Burkholderia cepacia 3 Ala Val Asp Asp Tyr AlaAla Thr Arg Tyr Pro Ile Ile Leu Val 1 5 10 15 4 44 DNA ArtificialSequence Description of Artificial Sequence Synthetic DNA 4 gcngtngaygactaygcngc nacncgntay ccnatyatnc tngt 44

1. An isolated esterase gene coding for an esterase capable of causingasymmetric hydrolysis of an organic carboxylic acid ester of acyclopentenolone of formula I:

wherein R₁ is hydrogen or methyl, and R₂ is C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₄ haloalkyl, a C₅-C₉ hydroxy aliphatichydrocarbon moiety which may be optionally protected on the terminalhydroxyl group thereof, or a C₅-C₉ fatty acid residue which may beoptionally protected on the terminal carboxyl group thereof, to producethe cyclopentenolone of formula I in (R)-form, and hybridizing to thebase sequence of SEQ ID NO:
 1. 2. The isolated esterase gene accordingto claim 1, wherein the homology of the gene to the base sequence of SEQID NO: 1 is 90% or higher.
 3. The isolated esterase gene according toclaim 1, having a base sequence coding for the amino acid sequence ofSEQ ID NO:
 2. 4. The isolated esterase gene according to claim 1, havingthe base sequence of SEQ ID NO:
 1. 5. A plasmid containing the esterasegene of claim 1, 2, 3 or
 4. 6. A transformant obtained by transformationwith the plasmid of claim
 5. 7. The transformant according to claim 6,which is a microorganism.
 8. An esterase produced by a microorganismhaving the esterase gene of claim 1, 2, 3 or
 4. 9. An esterase producedby the transformant of claim
 6. 10. A process for producing en esterase,which comprises cultivating the transformant of claim 6 to produce anesterase capable of causing asymmetric hydrolysis of an organiccarboxylic acid ester of a cyclopentenolone of formula I:

wherein R₁ is hydrogen or methyl, and R₂ is C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₄ haloalkyl, a C₅-C₉ hydroxy aliphatichydrocarbon moiety which may be optionally protected on the terminalhydroxyl group thereof, or a C₅-C₉ fatty acid residue which may beoptionally protected on the terminal carboxyl group thereof, to producethe cyclopentenolone of formula I in (R)-form.
 11. A method for theoptical resolution of a cyclopentenolone of formula I:

wherein R₁ is hydrogen or methyl, and R₂ is C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₄ haloalkyl, a C₅-C₉ aliphatic hydrocarbonmoiety which may be optionally protected on the terminal hydroxyl groupthereof, or a C₅-C₉ fatty acid residue which may be optionally protectedon the terminal carboxyl group thereof, which comprises allowing theesterase of claim 8 to act upon an organic carboxylic acid ester of thecyclopentenolone of formula I for asymmetric hydrolysis of the ester;and separating the cyclopentenolone of formula I in (R)-form from theester of the corresponding enantiomer thereof.
 12. The opticalresolution method according to claim 11, wherein the cyclopentenolone offormula I is 4-hydroxy-3-methyl-2-(2-propenyl)cyclopent-2-en-1-one. 13.The optical resolution method according to claim 11, wherein thecyclopentenolone of formula I is 4hydroxy-3-methyl-2-(2-propynyl)cyclopent-2n-1-one.
 14. An isolated esterasegene: (a) coding for an esterase capable of causing asymmetrichydrolysis of an organic carboxylic acid ester of cyclopentenolone offormula I:

wherein R₁ is hydrogen or methyl, and R₂ is C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₄ haloalkyl, a C₅-C₉ hydroxy aliphatichydrocarbon moiety which may be optionally protected on the terminalhydroxyl group thereof, or a C₅-C₉ fatty acid residue which may beoptionally protected on the terminal carboxyl group thereof, to producethe cyclopentenolone of formula I in (R)-form; (b) coding for apolypeptide having 363 amino acids; (c) having a recognition sequencefor the restriction endonuclease for SalI at a region of 537^(th) to542^(nd) in the said gene; and (d) having no recognition sequence forthe restriction endonuclease SmaI and XhoI.
 15. An isolated esterasegene: (a) coding for an esterase capable of causing asymmetrichydrolysis of an organic carboxylic acid ester of a cyclopentenolone offormula I:

wherein R₁ is hydrogen or methyl, and R₂ is C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₄ haloalkyl, a C₅-C₉ hydroxy aliphatichydrocarbon moiety which may be optionally protected on the terminalhydroxyl group thereof, or a C₅-C₉ fatty acid residue which may beoptionally protected on the terminal carboxyl group thereof, to producethe cyclopentenolone of formula I in (R)-form, wherein the isoelectricpoint of the esterase is 6.0; and (b) being contained in the depositedstrain FERM BP-5740.
 16. An isolated esterase gene according to claim 1,having: (a) a base sequence of SEQ TD NO: 1; or (b) a base sequencecoding for the amino acid sequence of SEQ ID NO:
 2. 17. An isolatedesterase gene: (a) coding for an esterase capable of causing asymmetrichydrolysis of an organic carboxylic acid ester of a cyclopentenolone offormula I:

wherein R₁ is hydrogen or methyl, and R₂ is C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₄ haloalkyl, a C₅-C₉ hydroxy aliphatichydrocarbon moiety which may be optionally protected on the terminalhydroxyl group thereof, or a C₅-C₉ fatty acid residue which may beoptionally protected on the terminal carboxyl group thereof, to producethe cyclonentenolone of formula I in (R)-form; and (b) having a basesequence coding for a region in SEQ ID NO: 2 of which N-terminal aminoacid sequence is the same as defined in SEQ ID NO: 3 which correspondsto 44^(th) to 58^(th) region in SEQ ID NO:
 2. 18. An isolated esterasegene: (a) coding for an esterase capable of causing asymmetrichydrolysis of an organic carboxylic acid ester of a cyclopentenolone offormula I:

wherein R₁ is hydrogen or methyls and R₂ is C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₄ haloalkyl, a C₅-C₉ hydroxy aliphatichydrocarbon moiety which may be optionally protected on the terminalhydroxyl group thereof, or a C₅-C₉ fatty acid residue which may beoptionally protected on the terminal carboxyl group thereof, to producethe cyclopentenolone of formula I in (R)-form, wherein the esterase isobtained by collecting bacterial cells of a harvested microorganism(FERMBP-5740).